JP4640742B2 - Method and means for producing a high-titer and safe recombinant lentiviral vector - Google Patents

Description

[0001]
Field of Invention
The present invention relates to novel lentiviral packaging vectors, transduction vectors carrying foreign genes of interest, stable packaging cell lines, stable producer cell lines, and their production for producing recombinant lentiviruses in mammalian cells. Regarding use.
Background of the Invention
Retroviral vectors are a common tool for gene delivery (Miller, Nature, 357: 455-460 (1992)). The ability of retroviral vectors to deliver unrearranged single copy genes to a wide range of rodent, primate and human somatic cells creates retroviral vectors well suited for gene transfer into cells.
[0002]
A useful accessory for creating recombinant retroviral vectors is a packaging cell line that supplies the proteins required for the production of infectious virions in trans, but such cells are endogenous. Cannot be packaged (Watanabe and Temin, Molec. Cell Biol., 3 (12): 2241-2249 (1983); Mann et al., Cell, 33: 153-159 (1983) Embretson and Temin, J. Viol., 61 (9): 2675-2683 (1987)). Considerations for the construction of retroviral packaging cell lines include rodents (Cloyd et al., J. Exp. Med., 151: 542-552 (1980)) and primates (Donahue et al., J. Exp. Med., 176 : 1125-1135 (1992)), production of high titer vector supernatants free of recombinant replication competent retrovirus (RCR), which has been shown to produce T lymphocytes.
[0003]
One way to minimize the possibility of RCR generation in packaging cells is to use a packaging function that expresses two genomes, eg, one that expresses the gag and pol gene products and the other that expresses the env gene product. (Bosselman et al., Molec. Cell Biol., 7 (5): 1797-1806 (1987); Markowitz et al., J. Virol., 62 (4): 1120-1124 (1988); Danos and Mulligan, Proc. Natl. Acad. Sci., 85: 6460-6464 (1988)). This method highlights the recombination frequency due to the ability of simultaneous packaging and subsequent transfer of these two genomes, as well as the presence of three retroviral genomes in the packaging cell to produce RCR. Minimize the ability to decrease.
[0004]
In the event that a recombinant occurs, mutations (Danos and Mulligan, supra) or deletions (Boselman et al., Supra) or Markowitz et al. Can be designed. In addition, deletion of the 3 'LTR on both packaging constructs also reduces the ability to form functional recombinants.
[0005]
Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the general retroviral genes gag, pol and env. The high degree of complexity allows the lentivirus to modulate its life cycle during the latent infection period.
A typical lentivirus is the human immunodeficiency virus (HIV), a pathogen of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages. In vitro, HIV can infect HeLa-Cd4 cells or T lymphocyte cells whose cell cycle has been suppressed by primary culture of monocyte-derived macrophages (MDM) and further by treatment with aphidicolin or γ-irradiation. it can.
[0006]
Cell infection depends on active transport of the pre-integration complex of HIV into the nucleus through the nuclear pore of the target cell. This is caused by the mechanism of transport of the target cell into the nucleus and the interaction of multiple partially overlapping molecular determinants in the complex. The determinants identified are the functional nuclear localization signal (NLS) of the C-terminal phosphotyrosine residue of the gag matrix (MA) protein, the karyophilic virion-related protein, vpr, and the gag MA protein including.
Summary of the Invention
Thus, the present invention provides a novel safe lentiviral vector related to the synthesis of both lentiviral vector transcripts that can be packaged and lentiviral proteins for the rapid production of high titer recombinant lentiviruses in mammalian cells. About. These results are infectious particles for delivering the foreign gene in question to target cells. The invention further provides cell lines for virus production.
Detailed Description of the Invention
The present invention provides a recombinant lentivirus capable of infecting non-dividing cells, as well as methods and means for producing the same. This virus is useful for the introduction and expression of nucleic acid sequences in vivo and ex vivo.
[0007]
The lentiviral genome and proviral DNA have three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeats (LTR). The gag gene encodes internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes RNA-dependent DNA polymerase (reverse transcriptase), protease and integrase; and the env gene Encodes the envelope glycoprotein. The 5 'and 3' LTRs can be used to promote virion RNA transcription and polyadenylation. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx (HIV-1, HIV-2 and / or SIV).
[0008]
Adjacent to the 5 'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and sequences necessary for effective encapsidation of viral RNA particles (psi site). This cis deficiency prevents encapsidation of genomic RNA if the sequences necessary for encapsidation (or packaging of retroviral RNA into infected virions) are lost from the viral genome. However, the resulting mutants can still direct the synthesis of all virion proteins.
[0009]
The present invention is capable of infecting non-dividing cells, including transfection into suitable host cells with two or more vectors having packaging functions, i.e. gag, pol and env, as well as rev and tat. A method for producing a recombinant lentivirus is provided. As described below, vectors lacking a functional tat gene are desirable for specific applications. Thus, to produce a packaging cell, for example, the first vector can provide a nucleic acid encoding viral gag and viral pol, and another vector encodes viral env. Nucleic acid can be provided. Incorporation of a vector providing a heterologous gene identified herein as a transfer vector into a packaging cell results in a producer cell that releases infectious viral particles that carry the foreign gene of interest.
[0010]
The aforementioned vectors themselves are outside the scope of the newly constructed vectors disclosed herein, but this is known in the art and is described in Naldini et al. (Sci., 272: 263-267). (1996)); and Zufferey et al. (Nat. Biotech., 15: 871-875 (1997)). In general, these vectors are plasmid-based or virus-based and are designed to retain sequences essential for the incorporation of foreign nucleic acids, selection of nucleic acids, and introduction into host cells. The gag, pol and env genes of the vector in question are also known in the art. The relevant gene is therefore cloned into the selected vector and then used for transformation of the target cell in question.
[0011]
In accordance with the vector and foreign gene design described above, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from any virus, including retroviruses. Preferably env is an amphotropic envelope protein that allows transduction of cells of human and other species.
[0012]
It is desirable to target the recombinant virus with a specific ligand for binding of this envelope protein to an antibody or targeting to a receptor of a specific cell type. By inserting the sequence of interest (including regulatory regions) into the viral vector, eg, with other genes encoding receptor ligands on specific target cells, the vector becomes target specific. Retroviral vectors can be made target specific, for example, by insertion of glycolipids or proteins. Targeting is accomplished by using an antigen-binding moiety such as an antibody or a recombinant antibody-type molecule, such as a single chain antibody, and often targets retroviral vectors. Those skilled in the art will know specific methods for achieving delivery of retroviral vectors to specific targets or will readily ascertain without undue experimentation.
[0013]
Examples of retrovirus-derived env genes include Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse breast cancer virus (MuMTV or MMTV), Gibbon monkey leukemia virus (GaLV or GALV), human Including but not limited to immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). Other env genes such as vesicular stomatitis virus (VSV) protein G (VSVG), hepatitis virus and influenza can also be used.
[0014]
Vectors that provide viral env nucleic acid sequences are operably linked to regulatory sequences such as promoters or enhancers. The regulatory sequence can be a eukaryotic promoter or enhancer, including, for example, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, the promoter-enhancer element is located within or adjacent to the LTR sequence.
[0015]
Preferably, the regulatory sequence is not endogenous to the lentivirus from which the vector was constructed. Thus, if the vector is generated from SIV, the SIV regulatory sequences found in the SIV LTR will be replaced by regulatory elements that do not originate from SIV.
VSV G is a desirable env gene because VSV G imparts a broad host cell range to recombinant viruses, but VSV G can be detrimental to host cells. Therefore, if a gene such as the gene for VSV G is used, an inducible promoter system is used, so that if VSV G expression is not required, VSV G expression is regulated to minimize host toxicity. It is preferable to be able to do this.
[0016]
For example, tetracycline was removed from the introduced cells using the tetracycline-regulated gene expression system described in Gossen and Bujard (Proc. Natl. Acad. Sci., 89: 5547-5551 (1992)). In some cases it can be used to provide inducible expression of VSV G. Thus, the tet / VP16 transactivator is present on the first vector and the sequence encoding VSV G is cloned downstream of the promoter controlled by the tet operator sequence on another vector.
[0017]
A transgene that is a heterologous or foreign nucleic acid sequence is operably linked to a regulatory nucleic acid sequence. As used herein, the term “heterologous” nucleic acid sequence means that the sequence originates from a foreign species or, if derived from the same species, is substantially modified from its original form. Alternatively, an unchanged nucleic acid sequence that is not normally expressed in a cell is a heterologous nucleic acid sequence.
[0018]
The term “operably linked” means that the regulatory sequence and the heterologous nucleic acid sequence are operably linked, resulting in the latter expression. Preferably, the heterologous sequence is linked to a promoter, resulting in a chimeric gene. This heterologous nucleic acid sequence is preferably under the control of either a viral LTR promoter-enhancer signal or an internal promoter, and the signal retained in the retroviral LTR can still result in effective expression of the transgene. .
[0019]
The foreign gene can be any of the nucleic acids in question that can be transcribed. In general, a foreign gene encodes a polypeptide. This polypeptide preferably has some therapeutic benefit. The polypeptide can compensate for the deficiency or absence of endogenous protein expression in the host cell. This polypeptide can confer new properties to the host cell, such as the chimeric signal receptor disclosed in US Pat. No. 5,359,046. Engineers can determine the suitability of foreign genes by practicing the techniques described herein and known in the art. For example, the engineer will know whether the foreign gene is of a size suitable for encapsidation and whether the foreign gene product is properly expressed.
[0020]
It is desirable to modulate the expression of genes that regulate molecules in cells by the incorporation of molecules by the methods of the present invention. The term “modulation” describes the suppression of gene expression when overexpressed or increased expression when underexpressed. Where cell proliferation disorders are associated with gene expression, nucleic acid sequences that interfere with gene expression at the translational level can be used. This method involves the transcription of specific mRNA, for example by using an antisense nucleic acid ribozyme or triplex agent to hide the mRNA with a nucleic acid or triplex material or cleaving it with a ribozyme. Alternatively, translation can be inhibited.
[0021]
An antisense nucleic acid is a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule (Weintraub, Sci. Am., 262: 40 (1990)). In the cell, the antisense nucleic acid hybridizes to the corresponding mRNA and forms a double-stranded molecule. Since cells do not translate double-stranded mRNA, antisense nucleic acids interfere with mRNA translation. Antisense oligomers of about 15 nucleotides or more are preferred because they are easily synthesized and are likely to cause less problems than larger molecules when incorporated into target cells. The use of antisense methods to inhibit in vitro translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem., 172: 289 (1988)).
[0022]
Antisense nucleic acids can be used to block the expression of mutant proteins such as amyloid precursor protein or dominant active gene products that accumulate in Alzheimer's disease. Such methods are also useful for the treatment of Huntington's disease, hereditary Parkinson's disease and other diseases. Antisense nucleic acids are also useful for inhibiting the expression of proteins associated with toxicity.
[0023]
The use of oligonucleotides to stop transcription can be by a mechanism known as a triplex strategy in which oligomers wrap around a double helix DNA and form a triple stranded helix. Thus, triplex compounds can be designed to recognize unique sites on selected genes (Maher et al., Antisense REs and Dev., 1 (3) 227 (1991); Helene, Anticancer Drug Dis ., 6 (6): 569 (1991)).
[0024]
Ribozymes are RNA molecules that have the ability to specifically cleave other single-stranded RNAs in a manner similar to DNA restriction endonucleases. Through modification of the nucleotide sequences encoding these RNAs, it is possible to manipulate molecules that recognize and cleave specific nucleotide sequences in RNA molecules (Cech, J. Amer. Med. Assn., 260: 3030). (1988)). The great advantage of such a method is that only mRNA with a specific sequence is inactivated.
[0025]
It is desirable to introduce a nucleic acid encoding a biological response modifier. Included in this category are immunostimulants that include nucleic acids encoding many cytokines that are classified as “interleukins”, such as interleukins 1-12. Also included in this category are interferons, particularly γ-interferon (γ-IFN), which do not necessarily act according to the same mechanism, but are tumor necrosis factor (TNF) and granulocyte-macrophage colony stimulating factor (GM- CSF). Desirably, such nucleic acids are delivered to bone marrow cells or macrophages to treat congenital enzyme deficiencies or immunodeficiencies. Nucleic acids encoding growth factors, toxic peptides, ligands, receptors or other physiologically important proteins can also be incorporated into specific non-dividing cells.
[0026]
Accordingly, the recombinant lentivirus of the present invention can be used to treat HIV-infected cells (eg, T cells or macrophages) with anti-HIV-molecules. In addition, for example, airway epithelium can be infected with a recombinant lentivirus of the invention having a cystic fibrosis transmembrane conductance regulatory (CFTR) gene for the treatment of cystic fibrosis.
[0027]
The methods of the present invention further include ex vivo transplantation of cells infected with the recombinant lentivirus of the present invention, or into the central nervous system or ventricular cavity or subdurally on the surface of the host brain. It is useful for transplantation or “grafting” of nerve cells, glial cells, fibroblasts or mesenchymal cells associated with infection in vivo. Methods for such grafting are well known to those skilled in the art and are described in Neural Grafting in the Mammalian CNS (1985) edited by Bjorklund and Stenevi.
[0028]
For diseases caused by protein product deficiencies, gene transfer can incorporate normal genes into infected tissues for compensation therapy, and even create animal models for diseases using antisense mutations. . For example, for infection of muscle, spleen or liver cells, it is desirable to insert a nucleic acid encoding factor VIII or IX into the lentivirus.
[0029]
The promoter sequence can be homologous or heterologous to the desired gene sequence. A wide range of promoters are available, including viral or mammalian promoters. Cell or tissue specific promoters can be used to target expression of gene sequences in specific cell populations. Mammalian or viral promoters suitable for the present invention are available in the art.
[0030]
Optionally, during the cloning step, a nucleic acid construct referred to as a transfer vector having a packaging signal and a heterologous cloning site further comprises a selectable marker gene. The marker gene is used to check the presence of the vector, so that infection or integration is confirmed. The presence of the marker gene ensures selection and growth of only host cells expressing the insert. Typical selection genes encode proteins that confer resistance to antibiotics or other toxicants such as histidinol, puromycin, hygromycin, neomycin, methotrexate, and cell surface markers.
[0031]
The recombinant virus of the present invention can introduce a nucleic acid sequence into mammalian cells. The term “nucleic acid sequence” means any nucleic acid molecule, particularly DNA, as discussed in detail herein. Nucleic acid molecules can be derived from a variety of sources including DNA, cDNA, synthetic DNA, RNA, or combinations thereof. Such nucleic acid sequences can include genomic DNA that may or may not include natural introns. Furthermore, such genomic DNA can be obtained in relation to the promoter region, poly A sequence or other related sequences. Genomic DNA can be extracted and purified from appropriate cells by means well known in the art. Alternatively, messenger RNA (mRNA) can be isolated from cells and used to make cDNA by reverse transcription or other means.
[0032]
Preferably, the recombinant lentivirus produced by the method of the present invention is a derivative of human immunodeficiency virus (HIV). This env will be derived from a virus other than HIV.
The methods of the present invention, in some embodiments, comprise three vectors that provide all the functions necessary for packaging of recombinant virions, such as gag, pol, env, tat and rev, as described above. provide. As noted herein, tat can functionally lack unexpected benefits. As long as these vectors are used for transformation and production of packaging cell lines and produce recombinant lentiviruses, there is no restriction on the number of vectors used.
[0033]
Such vectors are incorporated into packaging cell lines by transfection or infection. This packaging cell line produces viral particles containing the vector genome. Methods of transfection or infection are well known to those skilled in the art. After co-transfection of the packaging vector and the transfer vector into the packaging cell line, the recombinant virus is recovered from the culture medium and titered by conventional methods used by those skilled in the art.
[0034]
Therefore, this packaging construct is introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, usually with a dominant selectable marker such as neo, DHFR, Gln synthetase or ADA, and then Selection and clones can be isolated in the presence of an appropriate drug. These selectable marker genes can be physically linked to the packaging gene in the construct.
[0035]
Stable cell lines designed so that the packaging function is expressed by suitable packaging cells are known. See, for example, US Pat. No. 5,686,279 disclosing packaging cells; and Oray et al. (Proc. Natl. Acad. Sci., 93: 11400-11406 (1996)).
The Zufferey et al. Paper shows a lentiviral packaging plasmid lacking the 3 'sequence of pol containing the HIV-1 env gene. This construct contains the tat and rev sequences and the 3 'LTR is replaced with a poly A sequence. The 5 'LTR and psi sequences are replaced by alternative promoters such as those that are inducible. For example, a CMV promoter or a derivative thereof can be used.
[0036]
The packaging vector in question involves further modifying the packaging function to enhance lentiviral protein expression and enhance safety. For example, all HIV sequences upstream of gag can be removed. In addition, sequences downstream of env can be removed. Furthermore, a step of modifying the vector can be performed to enhance RNA splicing and translation.
[0037]
In order to provide a vector with less potential for the production of replication-competent lentiviruses, the present invention provides a functional deletion of the tat sequence, a regulatory protein that promotes viral expression by a transcriptional mechanism. Such lentiviral packaging plasmids are provided. As a result, the tat gene is partially or completely deleted, or various point mutations or other mutations occur in the tat sequence, rendering the gene non-functional. Engineers can practice known techniques for rendering the tat gene non-functional.
[0038]
The techniques used for vector construction and cell transfection and infection are widely practiced in the art. The practitioner is familiar with standard instrumental materials that describe specific conditions and procedures. However, guidelines are provided in the following paragraph for convenience.
Standard ligation and restriction methods well known in the art are used to construct the vectors of the invention (see Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1982). The isolated plasmid, DNA sequence or synthesized oligonucleotide is cleaved, adapted to the desired shape and religated.
[0039]
Site-specific DNA cleavage is performed by treatment with an appropriate restriction enzyme (or enzymes) under conditions understood in the art and, in particular, as specified by the manufacturer of a commercially available restriction enzyme. For example, see the New England Biolabs product catalog. In general, about 1 μg of plasmid or DNA sequence is cleaved with 1 unit of enzyme in about 20 μl of buffer solution. Typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of approximately 1-2 hours at approximately 37 ° C. are feasible, but variations thereof are acceptable. After each incubation, the protein is removed by extraction with phenol / chloroform, followed by extraction with ether, and the nucleic acid is recovered from the aqueous fraction by ethanol precipitation. If desired, size separation of the cleaved fragments can be performed by polyacrylamide gel or agarose gel electrophoresis using conventional methods. A general description of size separation can be found in Method of Enzymology, 65: 499-560 (1980).
[0040]
Restricted fragments were 50 mM Tris (pH 7.6), 50 mM NaCl, 6 mM MgCl.₂ Of E. coli DNA polymerase I in the presence of 4 deoxynucleotide triphosphates (dNTPs) in 5 mM, 10 mM DTT and 5-10 μM dNTPs using an incubation time of about 15-25 minutes at 20 ° C. It can be blunt ended by treatment with a large fragment (Klenow). The Klenow fragment fills the 5 'sticky end, but breaks the protruding 3' single strand even if four dNTPs are present. If desired, selective repair can be performed by supplying a single dNTP or a selected dNTP within the constraints indicated by the nature of the sticky ends. After treatment with Klenow, the mixture is extracted with phenol / chloroform and ethanol precipitated. Treatment with S1 nuclease or Bal-31 under appropriate conditions results in hydrolysis of any single stranded portion.
[0041]
The ligation can be performed in a volume of 15-50 μl under the following standard conditions and temperature: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mM DTT, 33 mg / ml BSA, 10 mM-50 mM NaCl, and 40 μM ATP, 0.01-0.02 (Weiss) units of T4 DNA ligase at 0 ° C. (ligation of “sticky ends”) or 1 mM ATP, 0.3-0.6 (Weiss ) Either at 14 ° C ("blunt end" ligation) using unit T4 DNA ligase. Intramolecular “sticky ends” are usually ligated at a total DNA concentration of 33-100 μg / ml (total end concentration of 5-100 mM). Intramolecular blunt end ligation is performed at a total end concentration of 1 μM (usually using a 10-30 fold molar excess of linker).
[0042]
Thus, in the present invention, lentiviral packaging vectors were determined by those skilled in the art as involving promoters and gag, pol, rev, env or combinations thereof, and the specific function or actual deletion of tat. Created to include other optional or necessary regulatory sequences and optionally other lentiviral accessory genes.
[0043]
A lentiviral transfer vector (Naldini et al., Supra; Proc. Natl. Acad. Sci., 93: 11382-11388 (1996)) is used to infect human cells with growth-arrested in vitro. And used to transduce nerves after direct injection into adult rat brain. This vector is efficient at the time of marker gene transfer into nerves in vivo, and long-term expression is achieved when there is no detectable pathology. The animals analyzed over the last 10 months after the single injection of the vector, the longest tested so far, showed no reduction in mean levels of transgene expression and no signs of histopathology or immune response ( Blomer et al., J. Virol., 71: 6641-6649 (1997)). Improved versions of lentiviral vectors have been developed in which the HIV virulence genes env, vif, vpr, vpu and nef have been deleted without compromising the ability of the vector to transduce non-dividing cells. Various attenuated versions have shown substantial improvement in the biosafety of the vector (Zufferey et al., Supra).
[0044]
In transduced cells, integrated lentiviral vectors generally have an LTR at each end. The 5 'LTR can cause accumulation of "viral" transcripts that can be recombinant substrates, especially in HIV-infected cells. The 3 'LTR promotes downstream transcription with risks resulting from activating cellular proto-oncogenes.
[0045]
The U3 sequence contains most of the HIV LTR. This U3 region contains enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation. Some of the promoter elements are essential for viral replication. Some of the enhancer elements are highly conserved in virus isolates and have been shown to be critical virulence factors for virulence. Enhancer elements can act to affect the replication rate in various target cells of the virus (Marthas et al., J. Viol., 67: 6047-6055 (1993)).
[0046]
Since viral transcription begins at the 3 'end of the U3 region of the 5' LTR, these sequences are not part of the viral mRNA and their copies from the 3 'LTR are both in the integrated provirus. Acts as a template for LTR generation. If the 3 'copy of the U3 region is altered in the retroviral vector construct, this vector RNA is still produced from the complete 5' LTR of the producer cell but cannot be regenerated in the target cell. Transduction of such vectors results in inactivation of both LTRs in the progeny virus. Thus, retroviruses are self-inactive (SIN) and these vectors are known as Sin transfer vectors.
[0047]
However, the degree of 3 'LTR deletion is limited. First, the 5 'end of the U3 region provides other essential functions in vector introduction that are necessary for integration (terminal dinucleotide + att sequence). Thus, the terminal dinucleotide and the att sequence can represent the 5 'boundary of the deleted U3 sequence. In addition, some ambiguously defined regions can affect the activity of polyadenylation sites downstream of the R region. Excessive deletion of the 3 'LTR U3 sequence may reduce polyadenylation of vector transcripts with deleterious consequences for both vector titer in producer cells and transgene expression in target cells. it can. On the other hand, limited deletions cannot abrogate LTR transcriptional activity in transduced cells.
[0048]
The new version of the lentiviral transfer vector described herein retains an increasing deletion of the U3 region of the 3 ′ LTR (FIG. 1: U3 from nucleotide -418 to the specified position of the U3 LTR). Deletion length: SIN-78, SIN-45, SIN-36 and SIN-18). Lentiviral vectors have been developed that have an almost complete deletion of the U3 sequence from the 3 'LTR without compromising both the titer of the vector in the producer cell and the expression of the transgene in the target cell. The most extensive deletions (-418 to -18) extend to the TATA box, which results in the inactivation of LTR transcriptional activity in the transduced cells. Therefore, the lower limit for 3 'deletion extends to the position containing the TATA box. This deletion is the remainder of the U3 region up to the R region. This means that the safety of the vector is dramatically increased. Various deletions were made by practices known in the art.
[0049]
Surprisingly, the average expression level of the transgene was higher even in cells transduced with the SIN vector compared to the more complete vector. This is probably due to the removal of transcriptional interference from the upstream HIV LTR on the internal promoter. Such SIN-type vectors with extensive deletions in the U3 region cannot be produced for retroviral vectors based on murine leukemia virus (MLV) without compromising transduction efficiency.
[0050]
The 5 'LTR of the transfer vector was modified by replacing some or all of the transcriptional regulatory elements in the U3 region with a heterologous enhancer / promoter. This change will enhance the expression of the transduced vector RNA in producer cells; to allow the generation of vectors in the absence of the HIV tat gene; and to “rescue” the aforementioned SIN vector It was created to remove a wild type copy of the upstream HIV LTR that could recombine with the 3 ′ deleted version.
[0051]
Therefore, the 5 ′ vector, which is a vector containing the above-mentioned changes in the 5 ′ LTR, can find use as an introduction vector for combination with a sequence that enhances expression and a packaging cell that does not express tat. .
Such 5 ′ vectors can also retain modifications in the 3 ′ LTR, as explained earlier, and can not only be used in packaging cells that do not simply enhance expression and do not express tat. This results in an improved transfer vector that can be further self-inactivated.
[0052]
Transcription from the HIV LTR is highly dependent on the transactivator function of the tat protein. In the presence of a tat often expressed by the core packaging construct present in the producer cell, transcription of the vector from the HIV LTR is strongly stimulated. Since full-length “viral” RNA has complete complementarity of the packaging signal, this RNA is efficiently encapsidated in vector particles and introduced into target cells. The amount of vector RNA available for producer cell packaging is the rate-limiting step in the production of infectious vectors.
[0053]
The enhancer region or enhancer and promoter region of the 5 'LTR was replaced by the human cytomegalovirus (CMV) or mouse rous sarcoma virus (RSV) enhancer or enhancer and promoter, respectively. See Figure 2 for an overview of the construct and hybrid vector code names. CCL and RRL vectors are completely substituted in the 5 'U3 region.
[0054]
A panel of control lentivector HR2 and 5 'hybrids were compared in producer cells transfected with the transfer vector in the presence or absence of packaging constructs providing tat transactivator. The transcription level of the four chimeric vectors is higher than that of the control lenti vector, both in the presence and absence of the packaging construct. All chimeric vectors efficiently introduce the transgene into the target cells, and the RRL vector acts like the control HR2 vector. Finally, integration of the vector into target cells was confirmed by examining cells transduced at early and late passages after transduction. There was no reduction in the ratio of transgene-positive cells indicating that the vector was integrated.
[0055]
The high level expression of the 5 'LTR-modified transfer vector RNA obtained in the producer cells in the absence of the packaging construct indicates that the resulting vector functions in the absence of the functional tat gene. Is shown. The loss of function of the tat gene previously shown for the packaging plasmid described herein results in a higher level of biosafety for Lentiantiviral vector systems conferred by many pathological activities associated with the tat protein. Will. Thus, a lentiviral vector with significantly improved biosafety is a SIN transfer vector that does not have a wild type copy of HIV LTR at either the 5 'end or the 3' end, which is described herein. Used in conjunction with tat-free packaging vectors.
[0056]
Viral supernatant is collected using conventional methods such as filtration of the supernatant 48 hours after transfection. The titer of the virus is 10⁶ NIH 3T3 cells or 10^Five It is measured in the presence of 8 μg / ml polybrene (Sigma Chemical Co., St. Louis, MO) by infection of individual HeLa cells. After 48 hours, transduction efficiency is assayed.
[0057]
Thus, the present invention provides methods and means for producing high titer recombinant viruses. Using such a viral particle preparation, target cells can be infected by methods known in the art. Thus, the present invention will find use in ex vivo gene therapy applications where target cells are removed from the host, transformed in the medium using known methods, and then returned to the host.
[0058]
The detailed description of the invention set forth below is a non-limiting example that illustrates various embodiments of the invention.
Example 1
Construction of lentiviral packaging plasmid
The lentiviral packaging plasmid was derived from the plasmid pCMVΔR8.9 (ΔVpr ΔVif ΔVpu ΔNef) previously described in the Zufferey et al. The remaining sequence of the nef gene of pCMVΔR8.9 was removed by digestion with XhoI and BstEII, filled with Klenow and religated. The construct was deleted for 100 base pairs and the HIV-1 truncated env reading frame was ligated to the polyadenylation site of genomic insulin, resulting in plasmid pCMVΔR8.73.
[0059]
In another embodiment of the invention, the plasmid pCMVΔR8.73 is deleted of 133 base pairs of the CMV-derived sequence downstream of the CMV promoter. This sequence contained a splice donor site, which was removed by digestion with SacII of plasmid pCMVΔR8.73 and religation into a larger fragment, resulting in plasmid pCMVΔR8.74.
[0060]
In another embodiment of the invention, all but the consensus 5 'splice donor site remaining in the plasmid pCMVΔR8.74 upstream of the start codon of the gag gene was removed. At the same time, the sequence upstream of the gag gene was changed with optimal translation efficiency to obtain plasmid pCMVΔR8.75. pCMVΔR8.75 was derived from pCMVΔR8.74 and was obtained by replacing the 94 base pair SstII-ClaI fragment with an SstII-ClaI oligonucleotide linker consisting of: 5'-GGGACTGGTGAGTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT-3 '1) And 5'-CGATCTAATTCTCCCCCGCTTAATACTGACGCTCTCGCACCCATGGCGGCGGCAGATCTCGAATTCACTCACCAGTCCCGC-3 '(SEQ ID NO:2).
[0061]
In another embodiment of the invention, the inducible packaging construct replaces the PstI-SacII fragment of pCMVΔR8.74 containing the CMV promoter with seven tandem copies of the tetracycline operator sequence linked to the minimal CMV promoter. Obtained. The tet-regulated packaging plasmid pTetΔR8.74 was obtained.
Example 2
Construction of lentiviral transfer vector
The lentiviral transfer vector plasmid was derived from the plasmid pHR'-CMV-LacZ previously described in Naldini et al. (Sci., 272: 263-267 (1996)). pHR2 is such that 124 base pairs of the nef sequence upstream of the 3 'LTR in pHR' are replaced with both polylinkers to reduce the HIV1 sequence and facilitate transgene cloning. Lentiviral transfer vector. pHR2 is derived from pHR'-CMV-LacZ, a 4.6 kb ClaI-StuI fragment was generated by PCR using pHR'-CMV-LacZ as a template and the following as an oligonucleotide with a 828 base pair ClaI- Obtained by replacing the StuI fragment with a 4.4 kb StuI-NcoI fragment and a 4.5 kb NcoI-ClaI fragment derived from pHR'-CMV-LacZ at three sites: 5'-CCATCGATCACGAGACTAGTCCTACGTATCCCCGGGGACGGGATCCGCGGAATTCCGTTTAAGAC-3 '(SEQ ID NO:3) And 5'-TTATAATGTCAAGGCCTCTC-3 '(SEQ ID NO:4).
[0062]
In another embodiment of the invention, pHR3 is a lentiviral vector in which 148 base pairs (including ATG) of the env coding sequence upstream of the Rev response element (RRE) of pHR2 are deleted. pHR3 was derived from pHR2 and was obtained by replacing the 839 base pair NotI-SpeI fragment of pHR2 with a 747 base pair NotI-SpeI fragment obtained by PCR using pHR2 as template and the following as oligonucleotide primers: : 5′-GCGGCCGCAGGAGCTTTGTTCCTTGG-3 ′ (SEQ ID NO:5) And 5'-TACGTAGGACTAGTCTCG-3 '(SEQ ID NO:6).
[0063]
In another embodiment of the invention, pRH5 is a lentiviral transfer vector in which the 310 base pair gag coding sequence (the entire gag coding sequence downstream of the 15th amino acid of the Gag protein) has been deleted from pHR2. pHR5 is digested with NruI of pHR2, NotI linker (synthetic oligonucleotide 5'-TTGCGGCCGCAA-3 ', (SEQ ID NO:7)), Digestion with NotI to excise a 310 base pair fragment, followed by religation.
[0064]
In another embodiment of the invention, pRH6 is a wrench in which the 5 ′ splice donor signal has been mutated (TGGT to TGAT) to enhance the production of full-length transcripts that can be packaged. Viral vector. pHR6 was derived from pHR5 and was obtained by replacing the 239 base pair AflII-ApoI fragment with a 239 base pair AflI-ApoI fragment generated by PCR using pHR2 as template and the following as oligonucleotide primers: 5 '-CCACTGCTTAAGCCT-3' (SEQ ID NO:8) And 5'-CAAAATTTTTGGCGTACTCATCAGTCGCCGCCCCTCG-3 '(SEQ ID NO:9).
[0065]
All PCR fragments were generated by first cloning the PCR reaction product directly into the TA cloning vector pCR2.1 (Invitrogen), followed by sequence verification and excision with the appropriate enzyme.
Example 3
Construction of 5 'LTR chimeric lentivirus vector
In another embodiment of the invention, the 5 'LTR of the lentiviral vector comprises an enhancer and promoter from the Rous sarcoma virus (RSV) U3 region linked to the R region of HIV-1 (plasmid pRRL). pRRL is a lentiviral transfer vector in which an RSV enhancer and promoter (nucleotides -233 to -1 relative to the transcription start position) are accurately fused to the R region of HIV-1 using an oligonucleotide linker. . pRRL refers to WO 97/07225, is derived from plasmids pRT43.RSV.F3 and pRH2, and the 3.4 kb EcoRI-HpaI fragment of pRT43.RSV.F3 is replaced with the 0.67 kb BglII-NotI fragment of pHR2. And a 1.7 kb NotI-StuI fragment of pHR2, 5′-AATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGGGTCTCTCTGGTTAGACCA-3 ′ (SEQ ID NO:10) And 5'-GATCTGGTCTAACCAGAGAGACCCGTTTATTGTATCGAGCTAGGCACTTAAATACAATATCTCTGCAATGCGGC-3 '(SEQ ID NO:11) Was replaced by a synthetic EcoRI-BglII oligonucleotide linker.
[0066]
In another embodiment of the present invention, the 5 ′ LTR of the lentiviral vector comprises Rous sarcoma virus (RSV) bound to the promoter region of HIV-1 (from −78 base pairs relative to the transcription start position). Contains an enhancer (nucleotides -233 to -50 relative to the transcription start position) (plasmid pRLL).
pRLL is a lentiviral transfer vector in which an RSV enhancer is fused to an HIV-1 promoter region using an oligonucleotide linker. pRRL was derived from plasmids pRT43.RSV.F3 and pHR2, and the 3.4 kb EcoRI-HpaI fragment of pRT43.RSV.F3 was replaced with the 0.724 kb AlwNI-NotI fragment of pRH2 and the 1.7 kb NotI-StuI fragment of pRH2. 5′-AATTGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATC-3 ′ (SEQ ID NO:12) And oligonucleotide 5'-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCC-3 '(SEQ ID NO:13) Was replaced by a synthetic EcoRI-AlwNI oligonucleotide linker.
[0067]
In another embodiment of the present invention (plasmid pCCL), the 5 ′ LTR of the lentiviral vector is an immediate early enhancer of human cytomegalovirus (CMV) bound to the R region of HIV-1 and Promoter (Boshart et al. (Cell, 41: 521-530 (1985)) contains the nucleotides -673 to -1 relative to the transcription start position). pCCL is derived from plasmids pRT43.2F3 (US Pat. No. 5,686,279) and pHR2, and the 3.8 kb SstI-HpaI fragment of pRT43.2F3 is replaced with the 1.7 kb BglII-NotI fragment of pRH2 and the 1.7 kb NotI-StuI fragment of pRH2. The oligonucleotide 5′-CGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCA-3 ′ (SEQ ID NO:14) And 5'-GATCTGGTCTAACCAGAGAGACCCCGGTTCACTAAACGAGCT-3 '(SEQ ID NO:15By replacing with a synthetic SstI-BglII oligonucleotide linker consisting of
[0068]
In another embodiment of the present invention (plasmid pCLL), the 5 ′ LTR of the lentiviral vector is a cytomegalovirus bound to the promoter region of HIV-1 (from −78 base pairs relative to the transcription start position) ( CMV) contains -220 to -673 enhancer nucleotides relative to the transcription start position. pCLL was derived from plasmids pRT43.2F3 and pHR2, and the 3.6 kb NcoI-HpaI fragment of pRT43.2F3 was replaced with the 0.724 kb AlwNI-NotI fragment of pRH2 and the 1.7 kb NotI-StuI fragment of pRH2, oligo-5'-CATGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGCCC -3 ′ (SEQ ID NO:16) And 5'-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTC-3 '(SEQ ID NO:17By replacement with a synthetic NcoI-AlwNI oligonucleotide linker.
Example 4
Construction of self-inactivating lentiviral vectors
pRRL.SIN-18 was derived from pRRL, and the 400 base pair EcoRV-PvuII fragment in the 3 'LRT was deleted by digestion and religation.
[0069]
pRRL.SIN-36 is derived from pRRL and a 493 base pair BbsI-AlwNI fragment of the 3'-side LTR was synthesized from a synthetic oligonucleotide 5'-GATATGATCAGATC-3 '(SEQ ID NO:18) And 5'-CTGATCA-3 'and a pRRL-derived 0.54 kb AlwN-AvrII fragment and a 6.1 kb AverII-BbsI fragment replaced by an oligonucleotide linker consisting of three sites.
[0070]
pRRL.SIN-45 is derived from pRRL, and the 3 'LTR 493 base pair BbsI-AlwNI fragment was synthesized from the synthetic oligonucleotide 5'-GATATGATCAGAGCCCTCAGATC-3' (SEQ ID NO:19) And 5'-CTGAGGGCTCTGATCA-3 '(SEQ ID NO:20) And a 6.1 kb AlwNI-AvrII fragment and a 6.1 kb AverII-BbsI fragment derived from pRRL.
[0071]
pRRL.SIN-78 is derived from pRRL, and the 493 base pair BbsI-AlwNI fragment of the 3 'LTR is converted into 5'-GATATGATCAGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATC-3' (SEQ ID NO:21) And oligonucleotide 5'-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCCTGATCA-3 '(SEQ ID NO:22) And an oligonucleotide linker consisting of a three-site ligation with a 0.54 kb AlwNI-AvrII fragment and a 6.1 kb AverII-BbsI fragment derived from pRRL.
Example 5
Construction of stable lentiviral packaging cells 00-28 and stable products of lentiviral vectors
Stable lentiviral packaging cells were generated using the 293G cell line. 293G cells were tet from the MD cassette (CMV promoter and intervention sequence from human β globin gene-exons 2 and 3, intron 2 and poly (A) sites).^R / VP16 transactivator and seven tetracycline operator sites (tet^O ) Expresses a minimal VSV envelope derived from the CMV promoter linked to the tandem repeats. Thus, the expression of VSV G is regulated by the tetracycline concentration in the culture medium and is suppressed in the presence of this antibiotic (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89: 5547-5551 ( 1992); Ory et al., Proc. Natl. Acad. Sci. USA, 93: 11400-11406 (1997)). 293G cells are usually maintained in DMEM / low concentration glucose culture medium supplemented with 10% donor bovine serum and containing 1 μg / ml tetracycline. 15 cm plates of 293G cells were transfected with Lipofectamine (GIBCO BRL) with 13.36 μg of packaging plasmid pCMVΔR8.74 and 1.33 μg of selection plasmid pZeoSV2. The medium was changed after 24 hours and after 48 hours the cells were divided into medium containing 250 μg / ml zeocin and 1 μg / ml tetracycline. After selection for 3-4 weeks, 250 clones were picked, transferred to 96-well plates, and the medium was screened for HIV-1 p24 Gag antigen by immunocapture using a commercially available kit. 52 p24 positive clones were grown for further analysis. The best 5 clones were determined to have a p24 value of 12-23 ng / ml. Four of these five clones were positive for VSV.G expression after tetracycline removal by Western blot analysis.
[0072]
Four additional p24 / VSV.G positive clones were analyzed for their ability to package lentiviral transfer vectors. These clones were cloned into a transiently generated lentiviral vector (VSV.G pseudotype) containing an A. Victoria green fluorescent protein (GFP) expression cassette activated by the CMV promoter with a multiplicity of infection of 10 and Infection was performed in the presence of polybrene (8 μg / ml). Infected clones were then expanded to remove tetracycline. At 72 hours after induction, the media was collected for 24 hours and the supernatant was filtered and snap frozen. Frozen supernatants were titrated on native HeLa cells for GFP gene transduction. By FACS analysis, the cell population generated by infection of packaging clone 00-28 (referred to as 10-28) has a maximum titer of 5 × 10^Four It was determined to have transducing units (T.U.) / Ml.
[0073]
Infected packaging population 10-28 was used to generate high titer producing clones of GFP lentiviral vector. 10-28 cells were examined by FACS and retained and expanded with the highest GFP expressing cells. This population was then infected four more times in succession ("ping") with transiently generated GFP lentivirus (VSV.G pseudotype). After each infection, supernatants were collected 72-96 hours after VSV.G induction. The supernatants were titered on HeLa cells and analyzed for p24 content by immunocapture assay. The infectious titer peaked after the third ping, 1.5 x 10⁶T.U./ml was reached (see FIG. 3). The cell population from the third ping was subcloned and the producer of the high titer vector was isolated.
[0074]
All articles and patent applications cited herein are hereby incorporated by reference in their entirety so that individual articles or patent applications are clearly and individually indicated to be incorporated by reference.
As will be apparent to those skilled in the art to which the present invention pertains, the present invention also encompasses transfection and transduction in forms other than those specifically shown above, such as transfection and transduction of other mammalian cell types. Or as long as they do not depart from the essential features. Accordingly, the specific embodiments of the present invention as described above are to be regarded as illustrative and not restrictive. The scope of the invention is, as noted, not limited to the embodiments limited in the foregoing description, but is the appended claims.
[Brief description of the drawings]
1 shows various lentiviral vectors. RSV is the Rous sarcoma virus enhancer / promoter; R is the R region of the LTR; U5 is the U5 region of the LTR; SD is the splice donor site, eg, the HIV 5 ′ major splice donor site: ψ is Psicapsidation signal sequence; Ga is part of the gag gene; RRE is the rev responsive element; SA is the splice acceptor sequence; U3 is the U3 region of the LTR.
FIG. 2 shows another lentiviral vector. CMV is a cytomegalovirus. In other respects, the symbols are the same as those shown in FIG.
FIG. 3 is a graph showing graded vector production with increasing amounts of transfer vector.
[Sequence Listing]

Claims (23)

Each is lentiviral transfer vector comprising a 5 'LTR and 3' LTR with the U3 region and the endogenous R region, part or all of the regulatory elements of the U3 region of the 5 'LTR is here the not endogenous to the lentivirus, are substituted by other regulatory elements that can be manipulated in mammalian cells, and the terminal dinucleotide and att sequences 5 'of the nucleotide bases of the U3 region of the side LTR' the 3 A lentiviral transfer vector in which everything except for is deleted.   The transfer vector according to claim 1, wherein the regulatory element that is not endogenous to the lentivirus is a cytomegalovirus enhancer, promoter, or enhancer and promoter.   The transfer vector according to claim 1, wherein the regulatory element that is not endogenous to the lentivirus is the rous sarcoma virus enhancer, promoter, or enhancer and promoter.   The transfer vector according to claim 1, wherein the vector carries a heterologous gene expressed.   The transfer vector according to claim 1, wherein the lentivirus is human immunodeficiency virus (HIV). 6. The transfer vector according to claim 5 , wherein the HIV is HIV-1. A lentivirus packaging plasmid, the lentivirus to lacking the sequence upstream of the gag gene which is endogenous, and lacking sequences downstream of the env gene that is endogenous to said lentivirus Where all of the sequences upstream of the gag gene that are endogenous to the lentivirus have been deleted and other sequences downstream of the nef and env genes that are endogenous to the lentivirus. A lentiviral packaging plasmid which is deleted and wherein the plasmid carries an expressed gag gene, an expressed pol gene, or an expressed gag and pol gene . The packaging plasmid of claim 7 , wherein the plasmid lacks a functional tat gene. The packaging plasmid according to claim 8 , wherein the tat gene is deleted. The packaging plasmid according to claim 8 , wherein the tat gene is mutated. 8. A packaging plasmid according to claim 7 , wherein the lentivirus is human immunodeficiency virus (HIV). 12. A packaging plasmid according to claim 11 , wherein the HIV is HIV-1. A method for producing a recombinant lentiviral vector comprising the following steps:
a) i) at least one lentivirus packaging plus Mi de of claim 7; and
ii) non-endogenous to said lentivirus which holds the env gene is expressed not endogenous to the lentivirus expression plasmid;
Transforming the cells to obtain packaging cells;
b) performing a plurality of transformations of the packaging cell with a lentiviral transfer vector carrying the expressed heterologous gene to obtain a producer cell;
c) culturing the producer cell in a medium; and
d) separating the producer cell from said medium, recovering said recombinant lentiviral vector from said medium. 14. The method of claim 13 , wherein the packaging cell lacks a functional tat gene. The lentiviral transfer vectors, each comprising a 5 'LTR and 3' LTR with the U3 region, wherein some or all of the regulatory elements of the U3 region of the 5 'LTR is the lentivirus 14. The method of claim 13 , wherein the method is replaced by other regulatory elements that are not endogenous to the cell and that can be manipulated in mammalian cells. The method of claim 15 , wherein each of the 5 ′ LTR and the 3 ′ LTR comprises an endogenous R region. 16. The method of claim 15 , wherein one or more nucleotide bases of the U3 region of the 3 ′ LTR are deleted. Are all deleted the U3 region other than 'the U3 region 5 of the side LTR' terminus dinucleotide and att sequence the 3 was The method of claim 17. A lentiviral vector system comprising the lentiviral packaging plasmid according to claim 7 and a lentiviral transfer vector, wherein the lentiviral transfer vector comprises a 5 'LTR and a 3' LTR each having a U3 region. wherein, where some or all of the regulatory elements of the U3 region of the 5 'LTR is not endogenous to the lentivirus, it is replaced by other regulatory elements that can be manipulated in mammalian cells, lentiviral Vector system. 20. The lentiviral vector system according to claim 19 , wherein the packaging plasmid lacks a functional tat gene. Wherein each of the 5 'LTR and the 3' LTR comprises endogenous R region, lentiviral vector system of claim 19, wherein. The lentiviral vector system according to claim 21 , wherein one or more nucleotide bases of the U3 region of the 3 'LTR are deleted. The deleted the 3 are all 'the U3 region of the side LTR 5' terminal dinucleotide and the U3 region other than the att sequences, lentiviral vector system of claim 22, wherein.

Images